LOCUS       sequence-458898-        5488 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration of msfGFP (or your Module Of Interest) in a random 
            fashion via Tn5 insertion. The antibiotic cassette (Gm) used for 
            selecting for the integration can be removed in a later stage with 
            pFNC..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5488)
  AUTHORS   Federici F, Luppino F, Aguilar-Vilar C, Mazaraki ME, Petersen LB, 
            Ahonen L, Nikel PI
  TITLE     CIFR (Clone-Integrate-Flip-out-Repeat): A toolset for iterative 
            genome and pathway engineering of Gram-negative bacteria.
  JOURNAL   Metab Eng. 2025 Jan 6;88:180-195. doi: 10.1016/j.ymben.2025.01.001.
  PUBMED    39778677
REFERENCE   2  (bases 1 to 5488)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Metab 
            Eng."; date: "2025-01-6"; volume: "88:180-195. doi"; pages: " 
            10.1016/j.ymben.2025.01.001"
COMMENT     Sequence Label: pCIFR-6
FEATURES             Location/Qualifiers
     source          1..5488
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      210..249
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     oriT            397..505
                     /note="incP origin of transfer"
     rep_origin      516..904
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(935..1021)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     misc_feature    1036..1054
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     RBS             1226..1234
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     protein_bind    2028..2075
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     promoter        join(2086..2091,2092..2108,2109..2114)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             2303..2836
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     protein_bind    2987..3024
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     misc_feature    3030..3048
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     terminator      3059..3153
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             3258..4685
                     /codon_start=1
                     /product="transposase from the bacterial Tn5 transposon 
                     (Reznikoff, 1993)"
                     /label=Tn5 transposase
                     /translation="MITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKS
                     ITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYR
                     HQVAEELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESG
                     KWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGL
                     YLYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNA
                     VLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGAGAER
                     QRMEEPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTP
                     DECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALWEGWEALQS
                     KLDGFLAAKDLMAQGIKI"
     promoter        4742..4833
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4834..4902,4903..5488,1..206)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 tgcgtagcgc gctgccggct ggctggttta ttgcggataa aagcggtgcg ggcgaacgtg
       61 gcagccgtgg cattattgcg gcgctgggcc cggatggtaa accgagccgt attgtggtga
      121 tttataccac cggcagccag gcgacgatgg atgaacgtaa ccgtcagatt gcggaaattg
      181 gcgcgagcct gattaaacat tggtaaaccg atacaattaa aggctccttt tggagccttt
      241 ttttttggac acgcgtttgt ccttttccgc tgcataaccc tgcttcgggg tcattatagc
      301 gattttttcg gtatatccat cctttttcgc acgatataca ggattttgcc aaagggttcg
      361 tgtagacttt ccttggtgta tccaacggcg tcagccgggc aggataggtg aagtaggccc
      421 acccgcgagc gggtgttcct tcttcactgt cccttattcg cacctggcgg tgctcaacgg
      481 gaatcctgct ctgcgaggct ggccgtaggc cggccgatct gaagatcagc agttcaacct
      541 gttgatagta cgtactaagc tctcatgttt cacgtactaa gctctcatgt ttaacgtact
      601 aagctctcat gtttaacgaa ctaaaccctc atggctaacg tactaagctc tcatggctaa
      661 cgtactaagc tctcatgttt cacgtactaa gctctcatgt ttgaacaata aaattaatat
      721 aaatcagcaa cttaaatagc ctctaaggtt ttaagtttta taagaaaaaa aagaatatat
      781 aaggctttta aagcctttaa ggtttaacgg ttgtggacaa caagccaggg atgtaacgca
      841 ctgagaagcc cttagagcct ctcaaagcaa ttttgagtga cacaggaaca cttaacggct
      901 gacatggggc gcgcccagct gtctagggcg gcggatttgt cctactcagg agagcgttca
      961 ccgacaaaca acagataaaa cgaaaggccc agtctttcga ctgagccttt cgttttattt
     1021 gatgccttta attaactgtc tcttatacac atctttgtgt ctcaggccgc ctaggccgcg
     1081 gccgcgcgaa ttcgagctcg gtacccgggg atccgcccat tgacaaggct ctcgcggcca
     1141 ggtataattg cacgagggcc caagttcact taaaaaggag atcaacaatg aaagcaattt
     1201 tcgtactgaa acatcttaat catgctaagg aggttttcta atgcgtaaag gtgaagaact
     1261 gttcaccggt gttgttccga tcctggttga actggatggt gatgttaacg gccacaaatt
     1321 ctctgttcgt ggtgaaggtg aaggtgatgc aaccaacggt aaactgaccc tgaaattcat
     1381 ctgcactacc ggtaaactgc cggttccatg gccgactctg gtgactaccc tgacctatgg
     1441 tgttcagtgt ttttctcgtt acccggatca catgaagcag catgatttct tcaaatctgc
     1501 aatgccggaa ggttatgtac aggagcgcac catttctttc aaagacgatg gcacctacaa
     1561 aacccgtgca gaggttaaat ttgaaggtga tactctggtg aaccgtattg aactgaaagg
     1621 cattgatttc aaagaggacg gcaacatcct gggccacaaa ctggaatata acttcaactc
     1681 ccataacgtt tacatcaccg cagacaaaca gaagaacggt atcaaagcta acttcaaaat
     1741 tcgccataac gttgaagacg gtagcgtaca gctggcggac cactaccagc agaacactcc
     1801 gatcggtgat ggtccggttc tgctgccgga taaccactac ctgtccaccc agtctaaact
     1861 gtccaaagac ccgaacgaaa agcgcgacca catggtgctg ctggagttcg ttactgccgc
     1921 aggtatcacg cacggcatgg atgaactcta caaataatct agagtcgacc tgcaggcatg
     1981 caagcttgcg gccgccaaag cccgccgaaa ggcgggcttt tctgtgtggt ttgtctggtc
     2041 aaccaccgcg gtctcagtgg tgtacggtac aaacccaatt taaatttgac ataagcctgt
     2101 tcggttcgta aactgtaatg caagtagcgt atgcgctcac gcaactggtc cagaaccttg
     2161 accgaacgca gcggtggtaa cggcgcagtg gcggttttca tggcttgtta tgactgtttt
     2221 tttgtacagc ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt
     2281 ttgatgttat ggagcagcaa cgatgttacg cagcagcaac gatgttacgc agcagggcag
     2341 tcgccctaaa acaaagttag gtggctcaag tatgggcatc attcgcacat gtaggctcgg
     2401 ccctgaccaa gtcaaatcca tgcgggctgc tcttgatctt ttcggtcgtg agttcggaga
     2461 cgtagccacc tactcccaac atcagccgga ctccgattac ctcgggaact tgctccgtag
     2521 taagacattc atcgcgcttg ctgccttcga ccaagaagcg gttgttggcg ctctcgcggc
     2581 ttacgttctg cccaagtttg agcagccgcg tagtgagatc tatatctatg atctcgcagt
     2641 ctccggagag caccggaggc agggcattgc caccgcgctc atcaatctcc tcaagcatga
     2701 ggccaacgcg cttggtgctt atgtgatcta cgtgcaagca gattacggtg acgatcccgc
     2761 agtggctctc tatacaaagt tgggcatacg ggaagaagtg atgcactttg atatcgaccc
     2821 aagtaccgcc acctaacaat tcgttcaagc cgagatcggc ttcccggccg cggagttgtt
     2881 cggtaaattg gacaattgtc taattaattg cggaccctag aggtcccctt ttttatttta
     2941 aaaatttttt cacaaaacgg tttacaagca taaaatctct gaggccggct tgtcgacgac
     3001 ggcggtctcc gtcgtcagga tcatccggga gatgtgtata agagacagac tagtcttgga
     3061 ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc
     3121 ggttgccgcc gggcgttttt tattggtgag aatccagggg tcccctggtt taaactacac
     3181 aagtagcgtc ctgaacggaa cctttcccgt tttccagaat ctgatgttcc atgtgacctc
     3241 ctaacatggt aacgttcatg attaccagtg cactgcatcg tgcggcggat tgggcgaaaa
     3301 gcgtgttttc tagtgctgcg ctgggtgatc cgcgtcgtac cgcgcgtctg gtgaatgttg
     3361 cggcgcaact ggccaaatat agcggcaaaa gcattaccat tagcagcgaa ggcagcaaag
     3421 ccatgcagga aggcgcgtat cgttttattc gtaatccgaa cgtgagcgcg gaagcgattc
     3481 gtaaagcggg tgccatgcag accgtgaaac tggcccagga atttccggaa ctgctggcaa
     3541 ttgaagatac cacctctctg agctatcgtc atcaggtggc ggaagaactg ggcaaactgg
     3601 gtagcattca ggataaaagc cgtggttggt gggtgcatag cgtgctgctg ctggaagcga
     3661 ccacctttcg taccgtgggc ctgctgcatc aagaatggtg gatgcgtccg gatgatccgg
     3721 cggatgcgga tgaaaaagaa agcggcaaat ggctggccgc tgctgcaact tcgcgtctga
     3781 gaatgggcag catgatgagc aacgtgattg cggtgtgcga tcgtgaagcg gatattcatg
     3841 cgtatctgca agataaactg gcccataacg aacgttttgt ggtgcgtagc aaacatccgc
     3901 gtaaagatgt ggaaagcggc ctgtatctgt atgatcacct gaaaaaccag ccggaactgg
     3961 gcggctatca gattagcatt ccgcagaaag gcgtggtgga taaacgtggc aaacgtaaaa
     4021 accgtccggc gcgtaaagcg agcctgagcc tgcgtagcgg ccgtattacc ctgaaacagg
     4081 gcaacattac cctgaacgcg gtgctggccg aagaaattaa tccgccgaaa ggcgaaaccc
     4141 cgctgaaatg gctgctgctg accagcgagc cggtggaaag tctggcccaa gcgctgcgtg
     4201 tgattgatat ttatacccat cgttggcgca ttgaagaatt tcacaaagcg tggaaaacgg
     4261 gtgcgggtgc ggaacgtcag cgtatggaag aaccggataa cctggaacgt atggtgagca
     4321 ttctgagctt tgtggcggtg cgtctgctgc aactgcgtga atcttttact ccgccgcaag
     4381 cactgcgtgc gcagggcctg ctgaaagaag cggaacacgt tgaaagccag agcgcggaaa
     4441 ccgtgctgac cccggatgaa tgccaactgc tgggctatct ggataaaggc aaacgcaaac
     4501 gcaaagaaaa agcgggcagc ctgcaatggg cgtatatggc gattgcgcgt ctgggcggct
     4561 ttatggatag caaacgtacc ggcattgcga gctggggtgc gctgtgggaa ggttgggaag
     4621 cgctgcaaag caaactggat ggctttctgg ccgcgaaaga cctgatggcg cagggcatta
     4681 aaatctaatc ggcgatcgct agtagcccgc ctaatgagcg ggcttttttt taattcccct
     4741 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4801 taaatgcttc aataatattg aaaaaggaag agtatgagca ttcagcattt tcgtgtggcg
     4861 ctgattccgt tttttgcggc gttttgcctg ccggtgtttg cgcatccgga aaccctggtg
     4921 aaagtgaaag atgcggaaga tcaactgggt gcgcgcgtgg gctatattga actggatctg
     4981 aacagcggca aaattctgga atcttttcgt ccggaagaac gttttccgat gatgagcacc
     5041 tttaaagtgc tgctgtgcgg tgcggttctg agccgtgtgg atgcgggcca ggaacaactg
     5101 ggccgtcgta ttcattatag ccagaacgat ctggtggaat atagcccggt gaccgaaaaa
     5161 catctgaccg atggcatgac cgtgcgtgaa ctgtgcagcg cggcgattac catgagcgat
     5221 aacaccgcgg cgaacctgct gctgacgacc attggcggtc cgaaagaact gaccgcgttt
     5281 ctgcataaca tgggcgatca tgtgacccgt ctggatcgtt gggaaccgga actgaacgaa
     5341 gcgattccga acgatgaacg tgataccacc atgccggcag caatggcgac caccctgcgt
     5401 aaactgctga cgggtgagct gctgaccctg gcaagccgcc agcaactgat tgattggatg
     5461 gaagcggata aagtggcggg tccgctgc
//
